(335a) Direct Quantification of Deubiquitinating Enzyme Activity in Intact Cells Using a Protease Resistant, Cell Permeable, Peptide-Based Reporter

Tremendous attention has recently shifted towards the implementation of molecularly-targeted therapeutics in treatment of different cancers. In the case of multiple myeloma, proteasome inhibitors Bortezomib and Carfilzomib have gained clinical success and FDA approval. However, one major challenge facing cancer patients is the presence of drug-resistant tumor cells that is further complicated by the high patient-to-patient variability due to the heterogeneous, personalized nature of cancer. Thus, there is an essential need for sensitive, high throughput biochemical assays to determine whether patients would benefit from such molecularly-targeted treatments. Deubiquitinating enzymes (DUBs) are an essential part the ubiquitin-proteasome system (UPS), the primary cellular pathway for protein degradation. Recently, DUBs have been indicated to play an important role in multiple myeloma patients developing a resistance to proteasome-based therapeutics. The goal of this project was to synthesize a novel peptide-based reporter of DUB activity that is cell permeable, long-lived, fluorescent, and specific to DUBs. The reporter (termed RW-LRGG-AFC) consisted of a β-hairpin motif [RWVRVpGOWIRQ] that acts as both of cell penetrating peptide (CPP) and ‘protectide’. The β-hairpin was conjugated onto the N-terminus of a DUB substrate consisting of the final four amino acids on ubiquitin (LRGG) with a C-terminal fluorophore (AFC, 7-amino-4-trifluoromethylcoumarin) to provide a detectable metric of DUB activity. The RW-LRGG-AFC reporter was initially characterized in HeLa and OPM2 cell lysates and shown to exhibit superior kinetics when compared to the unprotected four amino acid peptide (Z-LRGG-AMC). After validating the performance in cell lysates, the RW-LRGG-AFC reporter was demonstrated to be internalized by intact HeLa and OPM2 cells allowing for direct visualization of DUB activity in both population-based studies by fluorometry and single cell-based studies by microscopy. Reporter performance was determined to be both time- and concentration dependent. Inhibitor experiments with PR-619 (a commercially available DUB inhibitor) were performed to determine the specificity of the reporter to DUBs as well as demonstrate its ability to detect DUB inhibition at the single cell level. Intracellular stability of the peptide was assessed using an established degradation assay in cell lysates followed by RP-HPLC analysis. Ultimately, the novel peptide-based reporter was observed to rapidly penetrate the cells and remain stable under cytosolic conditions during the course of several hours. This new biosensor allows for the dynamic measurement of DUB activity in intact cells and has the potential to be incorporated into high-throughput screening (HTS) platforms to allow for personalized diagnostics.